The overall goal of this research is to understand the mechanisms by which early life stress triggers long-lasting defects in intestinal epithelial barrie function. The proposed studies with investigate the interactive signaling between the enteric cholinergic nervous system, intestinal mast cells, and the intestinal epithelial barrier utilizing novel pig and murine model systems. The project, based on previous studies and recent preliminary data, will test the hypothesis, that early life stress-associated GI disease is caused by a multistep mechanism in which long-term sensitization and increased abundance of cholinergic enteric nerves triggers persistent mast cell activation and subsequent intestinal barrier dysfunction. Understanding the early life signaling pathways that trigger long-lasting intestinal barrier dysfunction has relevance understanding the development of important human disorders including the Inflammatory Bowel Diseases (IBDs) and Irritable Bowel Syndrome (IBS). At the same time, these studies will have significant relevance to understanding and treating intestinal disease of agricultural animals such as the pig which undergo early life production stressors that have long-lasting deleterious influence on disease resistance, growth rate, and feed efficiency throughout the animal's production lifespan. In Specific Aim 1, we will determine the mechanism by which early life stress triggers long-term amplification of enteric cholinergic nervous system activity. In Specific Aim 2, we will utilize innovative in vitro and in vivo model systems to unravel the mechanisms by which Ach triggers persistent intestinal mast cell activation. In Specific Aim 3, we seek to understand the impact of early life stress and cholinergic nerve-mast cell signaling on alterations in epithelial tight junction protein function and structure in porcine and human models. These specific aims are designed to gain a fundamental understanding of how early life experiences contribute to the development of GI disease in adult life and will bring to light opportunities to develop novel approaches for the prevention and treatment of important GI diseases in humans and agricultural animals.